The role of lipoxygenase (LO) activation and oxidative stress in atherosclerosis and vascular injury due to high glucose (HG) and insulin resistance will be tested in this project. Our studies have implicated the NO pathway in vascular smooth muscle cell (VSMC) dysfunction under diabetic conditions. We showed that HG, growth factors and cytokines could induce 12-LO activity and expression. LO products lead to growth and chemotactic effects in VSMC. In this renewal, we will focus on i) the cellular and molecular mechanisms by which HG and LO products mediate their effects in VSMC and ii) the in vivo relevance of 12-LO activation in animal models of diabetes and atherosclerosis and vascular injury by inducing the transcriptional regulation of key genes that mediate VSMC growth, migration and matrix remodeling. The main hypotheses is that the LO pathway can play a role in the pathogenesis of atherosclerosis and vascular injury by inducing the transcriptional regulation of key genes that mediate VSMC growth, migration and matrix remodeling. The Specific Aims are 1) To examine the signal transduction mechanisms of HG and LO product-mediated gene expression in porcine VSMC. Here we will examine the effects of HG and LO products on the activation of ERK1/2 and p38 mitogen-activated protein kinases. We will also examine the role of IkappaB kinases in HG and LO product-induced of NF-kappaB. The intermediary role of 12-LO will be tested with the aid of novel 12-LO ribozymes. 2) To examine the mechanisms of transcriptional regulation of key atherogenic genes by HG and LO products, and to determine the functional role of LO activation in HG and angiotensin II (Ang II) actions. We will examine the regulation of the inflammatory gene, VCAM-1, the matrix protein fibronectin, and the matrix regulating MT1-MMP. The functional relevance of 12-LO activation in vitro will be examine in VSMC that overexpress 12-LO cDNA or the 12-LO ribozyme. 3) To clarify the in vivo relevance of 12- LO and MAPK activation in the pathophysiology of diabetic vascular complications. We will evaluate histology, 12-LO expression, as well as 12 LO-regulated signals and genes in mouse models including leukocyte- type 12-LO knock-out mouse cross bred to the apoE and/or the diabetic background. We will examine whether 12-LO mediates the accelerated neointimal thickening in a rat model of insulin resistance and diabetes. A unique aspect of this project will be to evaluate vascular changes and the role of 12-LO in a porcine model of accelerated atherosclerosis due to diabetes. In this model we will test the effect of: 1) tight glucose control with insulin, 2) pharmacologic inhibition of lipid peroxides using a novel anti-inflammatory agent lisofylline 3) effects of coronary artery stenting. This project will utilize both Cores and closely interact with Projects 2-4. The results should increase our knowledge of the factors leading to accelerated cardiovascular disease in diabetes and lead to new therapeutic advances.